Persistent pain affects 100 million Americans and 15 million Ukrainians. The immune system critically contributes to pathogenesis of inflammatory and neuropathic pain, and precise understanding of mechanisms through which particular immune mediators contribute to sensitization of nociceptive neuronal pathways will be essential for developing more efficacious treatment strategies. The complement system is a principal component of innate immunity that contributes to host defenses via diverse mechanisms. In spite of growing evidence implicating the complement system in various chronic pain states, the underlying mechanisms are not well understood. Our main objectives for this collaborative proposal between the US and Ukrainian groups are to elucidate complement-dependent spinal mechanisms that contribute to the development of neuropathic pain, and at a broader level, to promote building and strengthening sustainable research capacity in Ukraine. Mechanical hypersensitivity and spontaneous pain are common features of neuropathic pain. The main nociceptive output pathway from the spinal cord to the brain underlying this abnormal pain processing is lamina I projection neurons (PNs) of dorsal horn (DH). Our patch-clamp recordings from these neurons using an innovative intact spinal cord preparation demonstrate abnormal regulation of spinal cord output following spared nerve injury (SNI), a common model of neuropathic pain that well reproduces many features of clinical neuropathic pain. Recent studies suggest that neuropathic pain is associated with a robust upregulation of complement effectors in the spinal cord, which ultimately leads to production of a highly active complement product, C5a. Intrathecal administration of C5a produces allodynia, whereas C5 knockout (KO) and C5a receptor (C5aR1) antagonists produce analgesic effects in animal models of neuropathic pain. Our preliminary data show that C5aR1 KO prevents mechanical hypersensitivity following SNI. C5aR1 in the DH is found primarily on microglia that is known to be activated in the DH after SNI. Moreover C5aR1 expression is increased after SNI. We will use a multidisciplinary approach including patch-clamp recordings, optogenetic stimulation and multi-photon Ca2+ imaging in innovative intact spinal cord preparation combined with behavioral pharmacology to test our central hypothesis that C5a/C5aR1 signaling plays important roles in neuropathic pain processing by impacting central sensitization via microglia-dependent signaling that enhances the output of lamina I PNs of the DH to the supraspinal structures. This proposal will provide mechanistic insight into the function of the complement system in the CNS pain processing, and may lead to the development of new analgesic drugs that target complement system. In its broader impact, this project will promote establishment of Center for Excellence in brain disorder research in Ukraine, and help attracting young Ukrainian scientists to this field, providing their training and advancing chronic pain research in this country.